The capacities to introduce a particular foreign or native gene sequence into a mammalian cell and to control the expression of that gene are of substantial value in the fields of medical and biological research. Such capacities provide a means for studying gene regulation, for defining the molecular basis of a disease, and for designing a therapeutic basis for the treatment of disease.
Gene transfer techniques can be used for two general purposes. First, they can be used to evaluate the regulation and function of a cloned gene following its modification and introduction into different cell types by studying, for example, the definition of the regulatory elements that control levels of gene expression, alternate modes of RNA splicing, post-translational processing of peptides, sorting of proteins to their appropriate cellular locations, and biological activities of proteins. Second, gene transfer techniques can be used to modify cells, such as those of the nervous system, in culture and in vivo. Such studies may, for example, involve analysis of cell lineage, alternation of phenotypic properties, and ablation of specific cell populations, as well as creation and correction of hereditary disease states. Gene transfer techniques can thus be used as a tool in understanding molecular aspects of the development, function, and survival of cells.
The introduction of a particular foreign or native gene into a mammalian host cell is facilitated by first introducing the gene sequence into a suitable nucleic acid vector. A variety of methods have been developed which are capable of permitting the introduction of such a recombinant vector into a desired host cell.
For example, such a recombinant vector can be introduced into the host cell by DNA-mediated transformation (Choo, K. H. et al., Gene 46:277-286 (1986); Perez, C. F. et al., Radiat. Res. 104:200-213 (1985); Horst, J. et al., Hoppey-Seyler's Z. Physiol. Chem. 363:445-448 (1982); Hirschhorn, R. R. et al., Fed. Proc. 41: Abstract 6525 (1982); Graham, F. L. et al., In: The Wistar Symposium Series, Volume 1 Introduction of Macromolecules into Viable Mammalian Cells, Alan R. Liss, Inc., New York, N.Y. page 3-26 (1980); Upcroft, P., Anal. Biochem. 162:1-4 (1987)). The vector can also be introduced into a mammalian cell by protoplast fusion (Yoakum, G. H., Biotechniques 2:24-26, 28-30 (1984)), or by micro-injection (Spandidos, D. A. et al., Eur. J. Cell. Biol. 37:234-239 (1985); Folger, K. R. et al. Molec. Cell. Biol. 2:1372-1387 (1982); Gordon, J. W. et al., Proc. Natl Acad. Sci. USA 77:7380-7384 (1980)). Unfortunately, the above-described techniques are relatively inefficient and unsuitable for use in situations which require that the recombinant molecule be introduced into all or most of the cells present in culture or in an animal.
Techniques of transgenic genetics have been used to achieve the efficient introduction of a cloned gene sequence into all or most of the cells of an animal. In such an approach, a recombinant plasmid is introduced into the pronuclei of a fertilized egg and permitted to develop into a transgenic animal. Such an animal will usually contain the introduced gene in all of the cells of its body including its germ line. Transgenic genetics is, however, a technically difficult and exacting procedure.
Viral vectors have been employed in order to increase the efficiency of introducing a recombinant vector into suitably sensitive host cells. Viruses which have been employed as vectors for the transduction and expression of exogenous genes in mammalian cells include SV40 virus (Chung, M. H. et al., Korean J. Microbiol. 25:165-172 (1987); Innis, J. W. et al., Molec. Cell. Biol. 3:2203-2210 (1983); Okayama, H. et al., Molec. Cell. Biol. 5:1136-1142 (1985)), bovine papilloma virus (Meneguzzi, G. et al., Embo. J. 3:365-372 (1984); Dimaio, D. et al., Proc. Natl. Acad. Sci. USA 79:4030-4034 (1982); Lusky, M. et al., Cell 36:391-402 (1984); Giri, I. et al. Virol. 127:385-396 (1983); Lusky, M. et al., Molec. Cell. Biol. 3:1108-1122 (1983)), etc.
Retroviruses which have been employed as vectors for the transduction and expression of exogenous genes in mammalian cells include the Moloney murine sarcoma virus (Perkins, A. S. et al., Molec. Cell. Biol. 3:1123-1132 (1983); Lee, W. H. et al., J. Virol. 44:401-412 (1982); Curran, T. et al., J. Virol. 44:674-682 (1982); Gazit, A. et al., J. Virol. 60:19-28 (1986)), etc. In contrast to methods which involve DNA transformation or transfection, the use of viral vectors can result in the rapid introduction of the recombinant molecule into a wide variety of host cells.
Efforts to introduce recombinant molecules into post-mitotic neurons and other neural cells have, however, been hampered by the inability of such cells to be infected by the above-described viral or retroviral vectors. Thus, the study of gene expression in neuronal cells, and the identification of therapies for treating neuronal disease have been hampered by the lack of suitable methods to accomplish gene transfer into neural cells. A need therefore exists for efficient viral vectors capable of mediating gene transfer into such cells.